Method for treating ischemic insult to neurons employing an ATP-sensitive potassium channel blocker

ABSTRACT

A method is provided for treatment of neuronal insult, such as caused by lack of oxygen, by administering an ATP-sensitive potassium channel blocker, such as a sulfonyl urea, for example, tolbutamide.

This is a continuation of application Ser. No. 556,52, filed Jul. 20,1990 abandoned.

FIELD OF THE INVENTION

The present invention relates to a method for treating neuronal insult,such as caused by lack of oxygen, in neurons prone to Parkinsoniandegeneration, by administering to a patient an ATP-sensitive potassiumchannel blocker.

BACKGROUND OF THE INVENTION

A species of potassium channel that is dependent on adenosinetriphosphate (ATP) was first described in cardiac muscle by Noma A.(1983), "ATP-regulated K⁺ channels in cardiac muscle," Nature 305:147-148. This channel has attracted increasing interest due to itsunusual and close association with cell metabolism. Ashcroft, F. M.(1988), "Adenosine 5-triphosphate-sensitive potassium channels," Ann.Rev. Neurosci. 11: 97-118. It is now well established that ATP-sensitivepotassium channels are present in diverse tissues i.e. cardiac muscle,(Kakei M. and Noma A. (1984) " Adenosine 5'-triphosphate-sensitivesingle potassium channel in the atrioventricular node cell of the rabbitheart," J. Physiol. 352: 265-284, Noma A. and Shibasake, T. (1985),"Membrane current through adenosine-triphosphate-regulated potassiumchannels in guinea-pig ventricular cells," J. Physiol. 363: 463-480),pancreatic beta cells (Findlay, I., Dunne, M. J., and Petersen, O. H.(1985a), "ATP-sensitive inward rectifier and voltage- and calciumactivated K⁺ channels in cultured pancreatic islet cells," J. Memb.Biol. 88: 165-172; Dunne, M. J., Findlay, I., Petersen, O. H. andWollheim, C. B. (1986), "ATP-sensitive K⁺ channels in aninsulin-secreting cell line are inhibited by D-glyceraldehyde andactivated by membrane permeabilization." J. Memb. Biol. 93: 271-279;Ashcroft, F. M. et al (1984), "Glucose induces closure of singlepotassium channels in isolated rat pancreatic β-cells," Nature 312:446-448); skeletal muscle (Sturgess, N. C., Ashford, M. L. J., Cook, D.L. and Hales, C. N. (1985), "The sulphonylurea receptor may be anATP-sensitive potassium channel," Lancet 8435: 474-475) and smoothmuscle (Standen, N. B., Quayle, J. M., Davies, N. W., Brayden, J. E.,Huang, Y. and Nelson, M. T. (1989), "Hyperpolarizing vasodilatorsactivate ATP-sensitive K⁺ channels in arterial muscle," Science 245:177-180). More recently, indirect evidence has suggested that theATP-sensitive channel may also be present in the brain: sulfonylureas,which are potent blocking agents of this channel in heart and betacells, display selective binding in certain brain regions (Mourre, C.,Ben Ari, Y., Bernardi, H., Fosset, M. and Lazdunski, M. (1989),"Antidiabetic sulfonylureas: localization of binding sites in the brainand effects on the hyperpolarization induced by anoxia in hippocampalslices," Brain Res. 486: 159-164) and indeed an endogenous ligand for acentral sulfonylurea receptor has been described (Virsolvy-Vergine, A.,Bruck, M., Dufour, M., Cauvin, A., Lupo, B. and Bataille, D. (1988), "Anendogenous ligand for the central sulfonylurea receptor," FEBS Letters242: 65-69). It has also been found that sulfonylurea binding sitesappear to be highest in regions of the brain associated with the controlof movement, i.e. motor cortex, cerebellar cortex, globus pallidus andsubstantia nigra (Mourre et al., supra, 1989).

Despite intensive research into the causes and cures for Parkinson'sdisease, the actual homeostatic mechanisms of physiological and indeedpathological neuronal regulation within the substantia nigra remainobscure In the brain, the substantia nigra has the highest density ofbinding sites for the sulphonylurea, glibenclamide (Mourre, C. et al,Brain Res. 486, 159-164 (1989)), a selective blocker of K_(ATP)(Sturgess, N., et al, Lancet ii 8453, 474-475 (1985); Schmid-Antomarchi,H., et al Biochem. Biophys. Res. Commun. 146, 21-25 (1987); and Weillede, J., et al, Proc. Natl. Acad. Sci. U.S.A. 85, 1312-1316 (1988)). Itis thus possible that in the substantia nigra this channel, which has anunusual and close association with cell metabolism (Ashcroft, F. M.,Rev. Neurosci. 11, 97-118 (1988)), may play a pivotal role in neuronalregulation.

DESCRIPTION OF THE INVENTION

It has now been found that the K_(ATP) channel is critical in theresponses of nigral neurons to changes in the neuronal microenvironment,for example, during ischaemia. It has further been found that ischaemiacauses the opening of potassium channels in a selective population ofneurons with distinct pharmacological and electrophysiologicalproperties. Under ischaemic conditions, these neurons are prone toParkinsonian degeneration.

Thus, in accordance with the present invention, a method is provided fortreating neuronal insult, in the brain, due to lack of oxygen, wherein atherapeutically effective amount of a pharmaceutical which blocks anATP-sensitive potassium channel in the brain is administered to amammalian species in need of such treatment.

In addition, in accordance with the present invention, a method isprovided for treating early stages of Parkinsoniandegeneration-compensation, as caused by ischemic insult to neurons proneto Parkinsonian degeneration, wherein a therapeutically effective amountof a pharmaceutical which blocks an ATP-sensitive potassium channel inthe brain is administered to a mammalian species in need of suchtreatment.

The ischaemic insult referred to above may result from lack of oxygen toneurons prone to Parkinsonian degeneration such as caused by exposure totoxic fumes, for example, as caused by pyridines or cyanide poisoning,which results in reduced oxygen consumption in the cell.

The pharmaceutical employed in the methods of the present invention willbe an effective blocker of the ATP-sensitive potassium channel in thebrain. Examples of such a pharmaceutical include, but are not limited tosulfonyl ureas such as glyburide(1-[[p-[2-(5-chloro-O-anisamido)-ethyl]phenyl]sulfonyl]-3-cyclohexylurea);chloropropamide(1-[(p-chlorophenyl)sulfonyl]-3-propylurea);glibenclamide;glipizide(1-cyclohexyl-3-[[p-[2-(5-methyl-pyrazinecarboximido)ethyl]phenyl]sulfonyl]-urea);tolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1-yl)amino]-carbonyl,-4-methyl),or tolbutamide (benzenesulfoamide,N-(butylamino)carbonyl]-4-methyl),with the latter being preferred. In addition, quinine may also beemployed in place of the sulfonyl urea.

Although the K-ATP channel blocker employed in the methods of theinvention may be administered systemically, such as orally orparenterally, it is preferred that the K-ATP channel blocker beadministered locally, for example, by carotid injection, lumbar punctureor cisternal puncture. The K-ATP blocker will be administered for aslong as a treatment for neuronal insult due to lack of oxygen ortreatment for early stages of Parkinsonian degeneration-compensation isrequired.

With regard to dosage of K-ATP channel blocker, where a wide region ofthe brain is to be treated, for example, by intracarotid injection,lumbar puncture or cisternal puncture, from about 0.1 to about 20mg/kg/treatment and preferably from about 0.5 to about 15mg/kg/treatment will be employed, depending upon the particular K-ATPchannel blocker employed.

Where the K-ATP channel blocker is to be administered sytemically, suchas orally or parenterally, it will be administered in an amount toachieve a steady state level of K-ATP channel blocker in the blood.Thus, for systemic treatment, the K-ATP channel blocker may beadministered in an amount within the range of from about 0.5 to about 20mg/kg for each treatment and preferably from about 1 to about 15 mg/kgfor each treatment.

In carrying out the method of the present invention, the K-ATP channelblocker may be administered to mammalian species, such as monkeys, dogs,cats, rats, and humans. The K-ATP channel blocker may be incorporated ina conventional systemic dosage form, such as a tablet, capsule, elixiror injectable. The above dosage forms will also include the necessarycarrier material, excipient, lubricant, buffer, antibacterial, bulkingagent (such as mannitol), anti-oxidants (ascorbic acid of sodiumbisulfite) or the like.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 to 3 are graphs or chart recordings which depict data obtainedin carrying out the experiment described in Example 5.

The following Examples represent preferred embodiments of the presentinvention.

EXAMPLE 1

An injectable solution for use in administering tolbutamide by injectionin the carotid artery or by lumbar puncture or cisternal puncture fortreating neuronal insult or early stages of Parkinsoniandegeneration-compensation is produced as follows:

    ______________________________________                                        Tolbutamide             250 mg                                                Sodium chloride          25 mg                                                Polyethylene glycol 400  1.5 l                                                Water for injection qs.  5 l.                                                 ______________________________________                                    

The tolbutamide and sodium chloride are dissolved in 1.5 liters ofpolyethylene glycol 400 and 3 liters of water for injection and then thevolume is brought up to 6.5 liters. The solution is filtered through asterile filter and aseptically filled into presterilized vials which arethen closed with presterilized rubber closures. Each vial contains 25 mlof solution in a concentration of 50 mg of active ingredient per ml ofsolution for injection.

EXAMPLE 2

An injectable for use in treating neuronal insult or early stages ofParkinsonian degeneration-compensation is prepared as described inExample 1 except that quinine is employed in place of tolbutamide.

EXAMPLE 3 AND 4

An injectable for use in treating neuronal insult or early stages ofParkinsonian degeneration-compensation is prepared as described inExample 1 except that glyburide or glipizide is employed in place oftolbutamide.

EXAMPLE 5

Recent evidence suggests that an APT-sensitive potassium channel ispresent in the brain. From ligand binding studies it has been inferredthat this relatively unfamiliar channel is particulary denselydistributed in areas associated with motor control. To ascertain therole of the ATP-K channel in the in vitro substantia nigra a model ofischaemia was used to lower intracellular ATP and the sulphonylureatolbutamide was used to assess the activation of ATP-K channels.

METHODS

Coronal slices of adult male guinea-pig mesencephalon were prepared asdescribed by Harris, Webb et al, Neurosci. 31, 363-730 (1989) with theexception that halothane was used as the anaesthetic. Slices wereperfused in the recording chamber with the following solution (in mM):NaCl 136, KCl 2, NaHCO₃ 26, KH₂ PO₄ 1.25, MgSO₄ 2, CaCl₂ 2.4, glucose10.7. Intracellular recordings were made in pars compacta neurons at thelevel of the mammillary bodies (Gustafson, E.L. et al, Brain Res. 491,297-306 (1989)), with microelectrodes filled with 3M potassium acetate(series resistance measured in csf 50-140 Mohm). Neurons werecharacterised by an ability to generate burst firing triggered from aslow inward current (`LTSgCa²⁺ `), activated by either a depolarizing orhyperpolarizing current pulse from a hyperpolarized membrane potential(Llinas et al, Brain Res., 294, 127-132 (1984), and Kita et al, BrainRes. 372, 21-30 (1986)). All drugs were applied via the perfusingsolution to effective concentrations stated. Tolbutamide was initiallyprepared as a 0.5M stock solution dissolved in DMSO.

FIG. 1

Action of cyanide and tolbutamide on membrane potential, inputresistance, firing frequency and generation of LTSgCa²⁺ evoked burstfiring. Cell characteristics:apparent membrane potential, -50 mV; inputresistance 113 Mohms; firing frequency, 28 Hz; exhibited LTSgCa²⁺. a:membrane response to injections of hyperpolarizing current. b:generation of LTSgCa²⁺ evoked burst firing, activated by depolarizingphase of a 0.6 nA hyperpolarizing pulse from a hyperpolarized membranepotential. c: control response prior to exposure to cyanide. d: in 100μM cyanide. e: in 200 μM cyanide, note change in input resistance,hyperpolarization and generation of LTSgCa²⁺. f: in 200 μM cyanide and50 μM tolbutamide. Hyperpolarizing pulse in C to F was 0.4 nA for 200ms.

FIG. 2

Chart record illustrating the time course of a typical experimentshowing the actions of cyanide on the electrophysiological properties ofa pars compacta neuron (same as in FIG. 1). Hyperpolarizing pulses (0.4nA, 200 ms) were given at 0.2 Hz. Bath applied cyanide (1-200 μM)induced dose-dependent decreases in firing frequency and inputresistance together with membrane hyperpolarization. The actions of 200μM cyanide were abolished by the presence of 50 μM tolbutamide in thebathing medium. Action potentials attenuated by frequency response ofchart recorder.

FIG. 3

Current-voltage relations of a nigrostriatal cell obtained prior to andduring exposure to cyanide. Cell characteristics: apparent membranepotential, -55 mV; input resistance, 113 Mohms; firing frequency, 2 Hz,exhibited LTSgCa²⁺ a membrane response to hyperpolarizing currentpulses, note time-dependent anomalous rectification. b: membraneresponse to hyperpolarizing current pulses in the presence of 400 μMcyanide. c: plot of current-voltage relations prior to (open circles)and during exposure to 400 μM cyanide. Each data point is average ofmeasurements made from 4 successive pulses, standard deviations lostwithin the symbol. Measurements made at 57 ms and 180 ms to allow foranomalous rectification. Lines fitted to data are simple regressionlines with correlation coefficients (r²) >0.997. The bathing mediumcontained 3.2 mM KCl at 33° C, assuming K⁺ _(i) =140 mM). Thecurrent-voltage plots interesect at -92 mV, this together with thedecrease in input resistance seen in the presence of cyanide suggeststhat cyanide increases membrane permeability to potassium ions.

Long-term recordings (30 min-4 hr) were made from a total of 30 cellslocated in the pars compacta region of the substantia nigra. Of these 18resembled nigral neurons described previously, 5. (Llinas, R., et al,supra; Kita, T., et al, supra; Nedergaard, S., et al, Exp. Brain Res.69, 444-448 (1988); Greenfield, S.A., et al, Exp. Brain Res. 70, 441-444(1988); Kapoor, R., et al, Exp. Brain Res. 74, 653-657 (1989); Harris,N. C., et al, Exp. Brain Res. 74, 411-416 (1989)), in that a slow inwardcurrent (`LTSgCa²⁺ `) (Llinas, R., et al, supra), could be triggered inthese neurons upon depolarization from a hyperpolarized restingpotential: this LTSgCa²⁺ in turn led to the generation of bursts ofaction potentials (FIG. 1b). Furthermore, this population of neurons wassensitive to application of the dopamine (DA) receptor competitiveantagonist, haloperidol (10-100 μM), which caused a decrease in restingpotential and/or an increase in firing rate (depolarization 11+/-4 mVstandard deviation (SD), 13 fold increase in firing rate, n=4).

When these neurons (n=15) were exposed to cyanide (1-400 μM), a markedresponse was seen (FIG. 2), consisting of a hyperpolarization (10+/-4 mVSD, n=15) accompanied by a decrease in input resistance (prior tocyanide 112+/-9.6 Mohms standard error of the mean (SEM), n=13; duringcyanide 84+/-7.7 Mohms SEM, n=13; P<0.001 paired Student's t-test) and adecrease in firing rate (prior cyanide 18+4.1 Hz SEM, n=12; duringcyanide 1.4+0.8 Hz SEM, n=12; P<0.001 paired Student's t-test) whichwere reversed as soon as the cyanide was withdrawn (FIG. 2). The actionof cyanide on the current-voltage relationship for these neuronssuggested that cyanide-induced hyperpolarization was attributable to theopening of potassium channels (FIG. 3, mean cyanide reversal potential,-96+/-4 mV SD, n=3). However, the effects of cyanide were completelyabolished in the presence of the sulphonylurea tolbutamide (50-100 μM),a selective blocker of K_(APT) channels (Trube, G., et al, PfluegersArch. 407, 493-499 (1986), Belles, B., et al, Pfluegers Arch. 409,582-588 (1987)), (n=6) (FIG. 1f and 2). Indeed, in the presence oftolbutamide, cyanide poisoning led to a small depolarization of theresting potential (FIG. 2). By contrast a second population of nigralneurons recorded in this study (n=12) did not display a LTSgCa²⁺, burstfiring, nor sensitivity to haloperidol (n=5). Furthermore, exposure tocyanide did not result in a hyperpolarization of the resting potentialof these cells (n=6).

The observation that cyanide-induced hyperpolarization only occurred ina certain population of nigral cells precludes a non-specific action atthe level of the membrane. Rather, the small depolarizations observed inthe presence of tolbutamide, or in the haloperidol-insensitive cellsduring cyanide application, is most simply attributable to a gradualimpairment of the ubiquitous sodium-potassium pump. The cells that werenormally hyperpolarized by cyanide were characterized by generation ofthe LTSgCa²⁺, has been shown to be indicative of long `apical` dendritesextending into the pars reticulata region of the substantia nigra(Harris et al, supra). It is these long dendrites which are known toprobably release DA (Glowindki, J., et al Chemical Neurotransmission(eds Stjarne, et al; Academic Press London) 245-299 (1981)). Inaddition, the excitation of these cells by haloperidol strongly suggeststhat they are under the tonic influence of endogenous dendritic DA (seeNedergaard et al, supra, and Kapoor et al, supra). This finding thusprompts the hypothesis that it is those nigral neurons controlled bydendritic DA which possess the means for a rapid response (ie theK_(ATP) channel) to changes in the extracellular melieu. Theobservations described here show that ischaemia in these neurons issensitively and selectively reflected in an opening oftolbutamide-sensitive potassium channels. Indeed, the cyanide inducedenhancement of the LTSgCa²⁺ seen in FIG. 1e, despite a fall in inputresistance would suggest a dendritic location for these channels on theapical dendrites, within the pars reticulata (Mourre et al, supra).

It should be noted that hyperpolarization deinactivates the LTSgCa²⁺which in turn leads to burst firing (Llinas et al, supra) and anon-linear increase in striated DA release (Gonon, G. G., Neurosci 10,333-348 (1988)). Hence in the cases of mild anoxia, the K_(ATP) couldinitiate a chain of events leading to functional neuronal compensation.This means of compensation for neuronal insult may be extended to thepre-symptomatic stages of Parkinsonism. Reduced oxygen consumption hasbeen implicated in nigral cell death (Sanchez-Ramos, J. R., et al,Progress in Parkinson's Research (eds Hefti, F. & Wiener, W. J.; PlenumPress) 145-152 (1988)), and indeed the resultant K_(ATP) mediatedhyperpolarization would mimic operationally the normal action of localendogenous DA (Nedergaard et al, supra). Hence via the sequence ofevents outlined above, levels of striatal DA release would remain thesame despite the reduced dendritic DA release in the pre-symptomatic yetpathological substantia nigra. It is possible therefore that thetolbutamide-sensitive potassium channel plays a vital role in bothphysiological regulation and pathological compensation in nigral cellscontrolled by local DA and hence analoguous to those prone toParkinsonian degeneration. However as the degeneration processprogressed, the resultant hyperpolarization via K-ATP channels would beso severe that firstly, the cell body as well as the dendrites would behyperpolarized, and hence activation of the LTSgCa²⁺ prevented (seeLlinas et al, 1984, supra); secondly, the very high levels ofextracellular K⁺ ions would lead to osmotic imbalance and hence evenfurther neuronal death.

SUMMARY

Ischaemia causes the opening of potassium channels in a selectivepopulation of neurons with distinct pharmacological andelectrophysiological properties. The response to ischaemia is abolishedby tolbutamide. These results suggest that an ATP-K channel sensitive toischaemia is present in the substantia nigra and furthemore it may playa pivitol role in normal and pathological mechanisms of homoeostasis. Inaddition, tolubutamide may be useful in treating insult to neurons proneto Parkinsonian degeneration.

What is claimed is:
 1. A method for treating neuronal insult in thesubstantia nigra of the brain due to lack of oxygen, which comprisesadministering to the substantia nigra of the brain of a mammalianspecies in need of treatment, a therapeutically effective amount of apharmaceutical which blocks an ATP-sensitive potassium channel in thesubstantia nigra of the brain and is effective in treating neuronalinsult in the brain due to lack of oxygen, where the pharmaceutical is asulfonyl urea which is tolbutamide, glyburide(1[p-2[5-chloro-O-anisamido)ethyl]phenyl]sulfonyl]-3-cyclohexyl-3-urea);chlopropamide (1-[[(p-chlorophenyl)sulfonyl]-3-propylurea;glipizide(1-cyclohexyl-3[[p-[2=(5-methylpyrazinecarboxamido)ethyl]phenyl]sulfonyl]urea);ortolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1yl)amino]carbonyl]-4-methyl).2. The method as defined in claim 1 wherein the sulfonyl urea istolbutamide.
 3. The method as defined in claim 1 wherein thepharmaceutical is administered by infusion into the substantia nigra andblocks the ATP-sensitive potassium channel.
 4. The method as defined inclaim 1 wherein the pharmaceutical is administered locally.
 5. Themethod as defined in claim 1 wherein the pharmaceutical is administeredlocally by injection in the carotid artery, lumbar puncture or cisternalpuncture.
 6. The method as defined in claim 1 wherein the pharmaceuticalis administered locally in an amount of from about 0.1 to about 20mg/kg/treatment.